Polymers are high molecular weight molecules, formed through the polymerization of one or more structural units, named monomers. Polymers formed by carbohydrate monomers monosaccharides) are referred to as polysaccharides. Smaller molecules (olygosaccharides) can be derived from the latter by chemical or enzymatic partial hydrolysis.
Chitin is a linear polysaccharide composed by D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) residues linked through β-(1-4) linkages (see schematics, where (1) and (2) represent GlcN and GlcNAc, respectively). Chitin molecules form intermolecular hydrogen bonds that result in three different crystalline forms, depending on their arrangement. α-chitin, which is the most common and stable form, is characterized by an antiparallel arrangement of the chains, while β-chitin is formed by parallel layers. The rarest form, γ-chitin, is characterized by an antiparallel and two parallel chains.

The hydrogen bonds are responsible for chitin's low solubility both in water and in most organic solvents. Acid or alkaline solutions cause polymer hydrolysis and deacetilation, and hence are not suitable for its solubilization. Hydrogen bonds are also responsible for the apparent absence of melting temperature, as well as for the polymer's high rigidity and low permeability of chitinous materials.
Chitin's physicochemical properties are also closely related to the proportion of the two structural monomers. When the molar fraction of GlcN in the polymer (referred as the degree of deacetilation, % DD) is higher than 50%, the polymer is named chitosan, chitin's main derivative. Due to its lower content in acetyl radicals, chitosan is soluble in weak acids, has a polyelectrolyte character and a higher reactivity. Chitosan is usually obtained by deacetilation of chitin.
Owing to these characteristics, chitosan has currently more applications than chitin. Its high specific binding capacity is used for the removal of oils, heavy metals, proteins and fine particulate matter from wastewaters (Hennen, 1996). The same property allows its use in affinity chromatography (Synowiecki et al., 2003) and for reducing cholesterol absorption. Chitosan goes through the human digestive track and, by binding to low density cholesterol, restricts its absorption into the blood stream (ICNHP, 1995; Hennen, 1996).
In the food industry, chitosan is mainly used for egg and fruit coating (Hennen, 1996; Kim et al., 2007), acting as a barrier for carbon dioxide and pathogenic microorganisms, thus increasing the food products life time. It is also used as an emulsifier, and a preserving and clarifier agent for beverages (Kim, 2004; Synowiecki et al., 2003). Chitosan is also used in cosmetics, namely in hair products, because of its high stability and lower electrostatic character (Kim, 2004).
Chitosan biomedical applications have become increasingly more relevant due to the biocompatibility and biodegradability of its derivatives that allows for its use in wound healing (Hamliyn et al., 2004; Tanabe et al., 2006; Singh et al., 2000), tissue scaffolds (Tangsadthakun et al., 2007), drug release systems (Singh et al., 2000), among others.
Chitin main applications include its use as suture material (Hamliyn et al., 2004; Okada et al., 2000; Singh et al., 2000), as antigen in animals infected by bacteria or fungi (Singh et al., 2000) or chitinase production enhancer in soils contaminated by organisms that have chitin in their cell walls (Okada et al., 1999; Hallman et al., 1999). It is also used for the manufacture of breathable textiles, such as socks (ICNHP, 1995).
In contrast to synthetic polymers, chitin and chitosan are biodegradable, which makes their use an environmental benefit.
Besides chitosan, chitin derivatives include polysaccharides in which the GlcNAc C6 hydroxyl group is substituted by other radicals, such as, for example, alkyl or carboxyl groups. The insertion of these new radicals increases the polymers functionalities, thus making it possible to develop new applications, such as new fibers, gels, etc.
Next to cellulose, chitin is the second most abundant biopolymer in Nature. It is mainly found in the cuticle and exoskeleton of organisms of the Phylum Arthropoda and Crustacea, and in the cell wall of yeasts and fungi. In those organisms, chitin renders the cells rigidity and mechanical strength, and plays an important role during meiosis (Keller et al., 1970; Momany et al., 1997). The presence of chitin or any of its derivatives has not yet been detected in bacteria nor in Myxomycetes fungi. Chitin extracted from crustaceous and arthropods is more rigid and has a higher degree of deacetylation than microbial chitin.
Most of the chitin and chitin derivatives commercially available is obtained from the shells of crustacean, such as crabs, shrimps and lobster. The extraction process usually includes three steps: demineralization, protein and lipid removal, and bleaching. The first step is performed by mixing the shells with acid (usually, HCl), while protein and lipid removal is carried out in an alkaline medium (NaOH or KOH) in the presence of ethanol.
Pigment removal (especially carotenoids) is achieved by washing with organic solvents, such as acetone, chloroform or mixtures of ethanol with ethers.
Nevertheless, the seasonal character of this raw material and the variability of the composition of the shells as a function of the species and age of the animal, makes this process rather expensive and with low reproducibility. The rigidity of the exoskeletons of species such as lobsters and crabs also difficults the extraction and makes it more expensive. Moreover, since the chitin extracted from the shells of crustacean is of animal source, its use for pharmaceutical and biomedical applications is highly restricted by the food and drugs administration (FDA) regulations. Also the presence of proteins and pollutants absorbed by the animal also difficults chitin purification. Due to the possibility of allergenic reactions, polymers extracted from crustacean are less suitable for biomedical applications.
While in arthropods and crustacean chitin is aggregated to protein and minerals of the shells (mainly calcium salts), in microorganisms it is associated to other cell wall polysaccharides. Its reductive chain terminations are linked to the non-reductive ends of β-(1,3)-glucans (glucose polymers), which are linked to β-(1,6)-glucans, galactomannans (polymers formed by galactose and mannose residues) and glycoproteins. The cell wall composition is strain dependent and it is also variable during the organism's growth phase. Changing the fermentation conditions, such as medium composition and concentration (e.g. substrate availability), temperature or dissolved oxygen concentration (Aguilar-Uscanga et al., 2003), may result in variation of the relative proportion of each of the cell wall components.
The microbial production of chitin allows for the use of inexpensive raw materials, with quasi unrestricted availability, and for the continuous optimization of the process. The adaptation of the cell wall to environmental conditions can be used with advantage for optimizing the process. In fact, it has already been demonstrated that chitin production by fermentation may be enhanced by the supplementation of the medium with specific ions and precursors for the enzymatic chitin synthesis (Camargo et al., 1967; Keller et al., 1970). Both the composition and the properties of the polymers are also more stable than the ones obtained by the traditional extraction method from crustacean.
Currently, there is no optimized protocol for the production of chitin using microorganisms, except when genetic manipulated organisms are used (Hammer et al., 2006). Most of the microbial chitin commercially available is extracted from Saccharomyces carlsbergensis from the beer industry or Aspergillus niger from the citric acid production (Versali et al., 2003). In the later, chitin may account for up to 42% of the microorganism's cell wall. Nevertheless, submerged Aspergillus niger cultures do not attain cell densities as high as those reached by some yeasts. On the other hand, chitin production by Saccharomyces species does not go above 8% of the cell dry weight. Wastes from the culture of some edible mushrooms, such as Agaricus bisporus and A. campestris, may also be used (GB2259709), but the chitin content is not higher than 8% of the organism's dry weight.
Pichia pastoris is a Hemiascomycetes/Saccharomycetes yeast, commonly used for the expression of heterologous proteins. This species' main advantage over other microorganisms used for chitin production is the fact that it attains high cell densities during its fermentation on a wide variety of substrates, including glucose, methanol or raw glycerol while keeping a high percentage of chitin in its cell wall. Moreover, glycerol is a low cost byproduct from the biodiesel industry, available in large quantities and reported to be efficiently used for P. pastoris growth (çelik et al., 2008). Thus, the use of glycerol byproduct from the biodiesel industry for the production of chitin and chitosan by P. pastoris can be a process for its valorization. Moreover, the operating costs are reduced since it is not necessary to use high dissolved oxygen concentrations for the culture to grow.
At present, there are no available reports on the use of Pichia pastoris for the industrial production of the biopolymers that are the object of the present invention.